Metal-chelating ligands are useful in diagnostic medicine as contrast agents. X-ray imaging, radionuclide imaging, ultrasound imaging and magnetic resonance imaging can each be enhanced by the use of a metal atom bound to a chelating ligand. For example, a chelating ligand can become a radiopharmaceutical when it is prepared as a chelate complex with 99mTc, 111In, 67Ga, 140La, 169Yb, 68Ga, 90Y, 188Re, 153Sm or other radioactive metal ions. When a chelating ligand is complexed with the stable isotopes of the lanthanides, tantalum, bismuth or other elements with molecular weight higher than iodine, the resulting complex absorbs x-rays sufficiently to act as an x-ray contrast agent. In some cases, the agents that are useful in x-ray imaging absorb, reflect or scatter ultrasound radiation sufficiently to be used as an ultrasound agent. If a chelating ligand is complexed with a paramagnetic metal atom that has a symmetric electronic ground state (e.g., Gd+3, and octahedral Mn+2, Fe+3, Cr+3) the resulting complex will be useful as a spin relaxation catalyst that is used in magnetic resonance imaging (also known as NMR imaging) as a contrast agent. If a chelating agent is complexed with a paramagnetic metal atom that has an unsymmetrical electronic ground state (e.g., dysprosium(III), holmium(III) and erbium(III), the resulting complex will be useful as a chemical shift agent in magnetic resonance imaging or in magnetic resonance in vivo spectroscopy. In addition, any paramagnetic metal ion complex may be used as a contrast agent by virtue of its magnetic susceptibility as disclosed by Villringer et al. (Magnetic Resonance in Medicine, 6, 164-174, 1988).
The chelating ligands can also be bifunctional. That is, they can bind tightly to the metal ion forming a chelate while at the same time bearing a second functionality which confers upon it desirable chemical, physical and/or biological properties. Desirable physical properties of the chelator differ depending on the diagnostic or therapeutic purpose of the metal chelate. Desirable physical properties common to all uses are high affinity for the metal ion bound to the chelator, and ease of synthesis. When it is desired to use the metal chelate as a contrast medium for NMR imaging or general purpose x-ray imaging, the desirable physical properties are high water solubility, high chemical stability and viscosity and osmolality of a formulated drug solution as close as possible to those of human blood. Further, in the specific instance of a spin relaxation catalyst, the greatest possible relaxivity is desired. Relaxivity as used herein is understood to be as the effectiveness, per mole of complex, of altering the relaxation times of the nuclei being imaged.
Human blood has an osmolality of 0.3 Osmol/kg-water. Hyperosmolality is a well known contributor to adverse patient reactions to injected contrast media, and the lower osmolality of newer x-ray agents is due to their being nonionic molecules (possessing a net zero overall charge) (Shehadi, W. H.; “Contrast media adverse reactions: occurrence, reoccurrence and distribution patterns”, Radiol, 1982, 143, 11-17. Bettman, M. A.; “Angiographic contrast agents; conventional and new media compared”, Am. J.Roentgen, 1982, 139, 787-794. Bettman, M. A. and Morris, T. W.; Recent advances in contrast agents, Radiol. Clin. North Am., 1986, 24, 347-357.). Many gadolinium-based NMR agents in the prior art that are useful have a net negative overall charge, and therefore their aqueous formulated solutions have high osmolality. For example, Gd(DTPA)2-where DTPA stands for diethylenetriaminepentaacetic acid is formulated for use at 0.5M in water as the N-methylglucamine salt. The osmolality of the solution is 1.6 to 2.0 Osmol/kg-water. New nonionic Gd complexes are described in U.S. Pat. Nos. 4,859,451 and 4,687,659. The preferred new gadolinium complexes of the present invention are nonionic—they are not salts. When these nonionic gadolinium complexes are formulated at 0.5M in water the osmolality of the solutions is 0.3-0.6 Osmol/kg-water. The complex should be generally inert to interaction with the body other than general tissue distribution and excretion, usually by the renal route, without, or minimally, depositing Gd metal in tissues for long periods of time. Gd complexes of macrocyclic aminocarboxylates are generally more chemically inert than Gd complexes of linear aminocarboxylates (P. Wedeking and M. Tweedle, Nucl. Med. Biol., 15, 395-402, 1988; M. Tweedle et al., Magn. Reson. Imog., 9, 409-415, 1991; and M. Tweedle, “Contrast and Contrast Agents in Magnetic Resonance Imaging”, edited by P. A. Rink, European Workshop on Magnetic Resonance in Medicine, 1989). The preferred aminocarboxylate ligands for Gd are therefore members of the macrocyclic aminocarboxylate class, and are, in addition, nonionic. These properties are important to NMR imaging, but, in addition, the effectiveness of an agent for NMR imaging can be increased by altering the chemical structure so as to increase the ability of the metal chelate to affect the relaxation times of water protons.
In radiopharmaceutical imaging the doses administered are relatively small so that matching the drug formulation's physical properties to those of human blood is relatively unimportant. In this use biological specificity is more important. In particular, one could use 99mTc as the metal and a chelating ligand which is functionalized with a biologically active entity such as a bile acid, fatty acid, amino acid, peptide, protein or one of numerous chemical entities known to bind receptors in vivo. NMR contrast media may also make use of biological specificity.
In radiopharmaceutical therapy, the metal ions may be chosen from among those known in the art; for example, 90Y, 188Re, 153Sm. For this purpose the chelating ligand is generally covalently bound to a disease specific entity such as monoclonal antibody. When the metal-chelator-antibody conjugate is injected into humans, it concentrates at the disease site, usually a malignant tumor. In this use the chelating ligand must contain a reactive functionality which allows for a covalent bond to be formed between the chelating ligand and the antibody. Important characteristics of the reactive functionality are as follows: (1) it must be covalently attached to the chelator such that it does not significantly diminish the affinity of the chelator for the metal ion; (2) it must allow simple synthesis in high yield of metal-chelator-antibody conjugates, the conjugate so-formed should have maximal affinity for its antigen, such affinity being minimally diminished as a result of covalently attaching the metal-chelator; (3) it should ideally allow for rapid excretion and/or optimal dosimetry of the radioactive metal chelator in the event that the metal-chelator-antibody conjugate is decomposed or metabolized in vivo.
When the metal is non-radioactive and paramagnetic such as gadolinium (III), the bifunctional chelate is useful in magnetic resonance imaging as a contrast agent, either as a discrete molecule or bound to substances such as lipids, sugars, alcohols, bile acids, fatty acids, receptor-binding ligands, amino acids, peptides, polypeptides, proteins, and monoclonal antibodies. When the metal is radioactive, such as yttrium(III) as 90Y, the bifunctional chelate is useful in labeling monoclonal antibodies for use in radiotherapy. When the metal is 99mTc, 111In, 201Tl, 67Ga, 68Ga or the like, the chelate is useful in radiopharmaceutical imaging.
Two general methods have been employed for making bifunctional chelates form chelating agents. In the first method one or more carboxylic acid groups of a polyaminopolycarboxylic acid chelator are activated by conversion to such activating groups as internal or mixed anhydrides, activated esters (e.g., p-nitrophenyl, N-hydroxysuccinimide, etc.) or with other derivatives known to those skilled in the art. The activated acid group is then added to the protein-chelator complex.
There are two problems with this method. First, using a potential donor group, the carboxylic acid, to react with the protein can diminish the strength of the chelate and contribute to the chemical lability of the metal ion. The second problem arises because the chelating ligands have several carboxylates that are not uniquely reactive. When the chelating ligand is combined with an activating agent more than one species can result because the number and chemical position of the groups activated cannot be adequately controlled. When a mixture of such variously activated chelating ligands is added to protein, protein-chelator complexes of variable and uncertain chelating strength can be formed. Also, multiple activation of carboxylic acids on a chelator leads to intra- and inter-molecular crosslinking which is a major source of decreased immunospecificity. This problem could be overcome by separating all of the products formed from the reaction of the activating agent with the chelating ligand, but that process is very laborious and makes the overall synthesis highly inefficient.
The second method for making a bifunctional chelate is to prepare a chelating ligand with a unique reactive function, such as an isothiocyanate, attached to the chelating ligand at a position that does not substantially diminish the strength with which the chelating ligand binds the metal ion. An article entitled “Synthesis of 1-(p-isothiocyanatobenzyl) derivatives of DTPA and EDTA, Antibody Labeling and Tumor-Imaging Studies” by Martin W. Brechbiel, Otto A. Gansow, Robert W. Atcher, Jeffrey Schlom, Jose Esteban, Diane E. Simpson, David Colcher, Inorganic Chemistry, 1986, 25, 2772 is illustrative of the above second method. Also, U.S. Pat. No. 4,885,363 describes these methods as they apply specifically to nonionic macrocyclic aminocarboxylates.
Wedeking et al., “Biodistribution and Excretion of New Gd-Complexes in Mice”, Abstracts of the 8th Annual Meeting of the Society of Magnetic Resonance in Medicine, 801, 1989, have disclosed the compound When used to chelate a paramagnetic ion, e.g., Gd, in magnetic resonance imaging, this compound was found to have poor water solubility, although acceptable relaxivity.